|Other Abstract||In recent years, crude oil pollution caused by natural or anthropogenic factors, has become increasingly serious, leading to long-term and devastating effects on the ecological system and human health. Bioremediation, using microorganisms to degrade crude oil, has been considered as an important environmental remediation technology, which is also more economical, effective, versatile and environmentally friendly than physical and chemical remediation. Particularly, the key of crude oil pollution bioremediation is to find microbial strains with high degradation activity, strong environmental adaptability and wide substrate range. In this study, Dietzia sp. CN-3 was isolated from the marine sediments of Penglai 19-3 platform in the Bohai Sea. D. sp. CN-3 had high petroleum degradation activity, wide substrate range and strong environmental adaptability, which could be potential in crude oil pollution bioremediation. Due to genomics and transcriptomics, the biological processes and metabolic pathways related to alkane degradation were predicted, especially to the alkane hydroxylase gene (alkB and CYP153). Furthermore, the function of alkB and CYP153 was identified through RT-qPCR, gene disruption and heterologous expression methods. Two bacterial strains of D. sp. CN-3 and Acinetobacter sp. HC8-3S, with sufficient laboratory research foundation, were functionally combined to construct a bacterial consortium, in order to provide theoretical basis and technical support for crude oil pollution bioremediation. The main contents and results were summarized as follows:
1. A highly efficient crude oil-degrading bacterium of D. sp. CN-3 was isolated from the marine sediments of Penglai 19-3 platform in the Bohai Sea. The strain degraded a wide variety of petroleum hydrocarbons, alkanes and aromatic hydrocarbons, including linear alkanes (C10-C36), branched alkanes (pristane and phytane), cycloalkanes and aromatic hydrocarbons (phenanthrene and pyrene). Besides, the strain had good tolerance to different temperature (4-42℃), pH (5-10) and salinity (0-160 g/L). Particularly, the petroleum hydrocarbon degradation activity was hardly influenced under high salinity conditions (85 g/L). In order to acceletate the petroleum hydrocarbons degradation, D. sp. CN-3 was able to produce surfactants or change cell surface hydrophobicity.
2. The complete genome sequence of D. sp. CN-3 was achieved, providing a comprehensive genetic background for different biological processes and metabolic pathways investigations. The genome contained a chromosome genome and two plasmid genomes, with total length of 3741379 bp and GC content of 70.66%. There were 3496 encoding genes, whose length accounted for 88.83% of the genome. The genome contained 50 tRNA, 9 rRNA and 12 gene islands. Large numbers of genes associated with alkane degradation were annotated, including one alkane hydroxylase gene alkB and one CYP153 of cytochrome P450 family, as well as variety of enzymes involved in alkane single-terminal oxidation pathway, such as alcohol dehydrogenase, aldehyde dehydrogenase and acyl-CoA synthetase, etc. In addition, 19 genes related to the aromatic compounds degradation were annotated in the genome, such as catechol 1,2-dioxygenase gene (catA), catechol 2,3-dioxygenase gene (catE), etc. It was speculated that the catechol pathway played a major role in aromatic compounds degradation by D. sp. CN-3, due to relatively complete genes in the genome.
3. Transcriptome sequencing of D. sp. CN-3 cultivated in sodium succinate and n-hexadecane cultures was completed to comprehensively analyze the genes expression in different biological processes. Transcriptome analysis indicated that the transcription profiles of CN-3 were significantly changed under n-hexadecane induction, and 546 differentially expressed genes (FDR<0.05 and |Log2FC|>1) were found, among which 168 genes were up-regulated and 378 genes were down-regulated. Many differentially expressed genes were related to lipid metabolism, amino acid metabolism, carbohydrate metabolism, xenobiotics biodegradation and metabolism, as well as transcription and translation. Potential new genes (31) were also found herein. In addition, alkB, CYP153, Fdx and FdR were strongly up-regulated (log2FC 5.3065-6.198), and genes related to the downstream metabolism of n-hexadecane were also up-regulated in different degrees, which could be speculated that CN-3 strain degraded n-hexadecane through the single-terminal oxidation pathway.
4. Taking advantage of in vitro and in vivo experiments, the molecular mechanism of alkB and CYP153 in division and cooperation of alkane degradation had been confirmed, which was considered as a potential new mechanism and quite different from previous reports. Through amplification and homology analysis of alkB and CYP153, it was found that there were four conserved motifs of Hist-1, Hist-2, Hist-3 and HYG-motif in the amino acid sequences, which were identified as AlkB alkane hydroxylase, and the CYP153 gene belonged to the CYP153A family of cytochrome P450. Using suicide plasmid of pK18, the gene disruption mutants of ΔalkB and ΔCYP were successfully constructed. It was confirmed that both alkB and CYP153 played important roles in the middle-chain alkanes, branched alkanes and long-chain alkanes degradation, but they had their own preferences. Specifically, CYP153 was mainly responsible for degradation of middle-chain alkanes and branched alkanes, and alkB was mainly responsible for long-chain alkanes degradation. There was a synergistic effect between alkB and CYP153 genes in C28 degradation. Meanwhile, these results were consistent with the transcriptional level analysis of alkB and CYP153 genes induced by different alkanes. From the perspective of synthetic biology, Pseudomonas putida F1 was used as the platform cell to introduce alkB gene to construct petroleum hydrocarbon-degrading cell factory. Consequently, P. putida F1 could degrade medium-chain and long-chain alkane (C14, C16, C24 and C26) and be potential in crude oil pollution bioremediation.
5. Relying on different petroleum degradation characteristics and biosurfactant production abilities, D. sp. CN-3 and Acinetobacter sp. HC8-3S, were functionally combined to construct a bacterial consortium. Compared to single strains, the petroleum degradation of this consortium was significantly enhanced, with 95.8% of degradation efficiency in 10 days, which degraded petroleum efficiently in the ranges of pH (4-10) and salinity (0-120 g/L). In the crude oil-contaminated soil microcosms, the degradation rate of bioaugmented treatment with the consortium was 485.8 mg kg-1 d-1, which was significantly higher than many previous reports, demonstrating the application potential of the consortium in crude oil pollution bioremediation.
In a summary, the petroleum hydrocarbon degradation performance and mechanisms, as well as alkane hydroxylase function in D. sp. CN-3 were investigated herein. The molecular mechanism of alkB and CYP153 in division and cooperation of alkane degradation had been confirmed to be a potential new one. This is valuable to provide references for mechanism research and molecular modification in other alkane-degrading strains, and provide theoretical basis and technical support for bioremediation of crude oil pollution.|